Gene therapy has been experimentally tried to clinically treat or prevent diseases, in particular genetic diseases. Gene therapy at the beginning has been studied using viruses as vectors, because the virus vectors were able to be introduced into cells with higher efficiency. After it was found that a plasmid DNA (pDNA) having an autonomously replicating ability could express gene information on the direct administration into muscle of animals, gene therapy using the plasmid DNAs has been investigated exclusively in light of the fact that pDNA possesses the higher safety, and may be more readily produced on a manufacturing scale than those of the virus vector.
Primary interests today in basic research for gene therapy using pDNAs are directed to an improvement in an introduction efficiency into cells, and an extension of a period of time during expression of gene information. For the purpose of the improvement in the introduction efficiency of pDNAs into cells, the method for encapsulating pDNA into a cationic liposome, and the method for preparing a complex with a polymer have been reported. For the purpose of the extension of the period during expression of gene information, the sustained-release formulation comprising collagen (Japanese Patent Publication (kokai) No. 71542/1997) or polyethylene vinyl acetate (Journal of Controlled release 47, 123 (1997)) as a carrier, each of which is highly biocompatible, has been reported. In practice, however, even if a gene formulation can serve these purposes, it would not make gene therapy widely prevalent unless the gene formulation can be produced commercially with a stability and an ability to maintain defined high qualities.
Biological activities of genes, active agents, in the gene formulations should be maintained securely during the preparation and the preservation of the formulations. It has been known that intra-muscular injection of closed circular pDNA which had been digested with a restriction enzyme results in an decreased gene information to the extent of about 10% of that provided by an inert closed circular pDNA. Consequently, in order to maintain the biological activities of genes it is important that a primary structure of pDNA should be retained all through the preparation and the preservation of the formulations, during which any unfavorable condition is expected.
Although some reports have discussed a stability of pDNA during the preservation in connection with the basic research of gene therapy as mentioned above [Proceedings of National Academy of Sciences of the USA, 93, 7305 (1996)], any systematical investigation about the preparation and the stability of gene formulations scarcely has been conducted yet. As shown in the test examples hereinafter, when the gene preparations comprising pDNA only, or comprising pDNA together with compounds useful for improving the introduction efficiency of pDNA or for extending the period of time during gene expression are exposed to lyophilization conditions usually employed in the formulation steps, or preservation conditions in which the qualities of the formulations would be securely maintained, an active agent (pDNA) is subject to degradation, and its biological activity is markedly impaired.